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- Fluoreszenz-Resonanz-Energie-Transfer (3)
- Charakterisierung (1)
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Long-range tertiary interactions between RNA tetraloops and their receptors stabilize the folding of ribosomal RNA and support the maturation of the ribosome. Here, we use FRET-assisted structure prediction to develop a structural model of the GAAA tetraloop receptor (TLR) interaction and its dynamics. We build the docked TLR de novo, label the RNA in silico and compute FRET histograms based on MD simulations. The predicted mean FRET efficiency is remarkably consistent with single-molecule experiments of the docked tetraloop. This hybrid approach of experiment and simulation will promote the elucidation of dynamic RNA tertiary contacts and accelerate the discovery of novel RNA and RNA-protein interactions as potential future drug targets.
We use machine learning for the selection and classification of single–molecule trajectories to replace commonly used user–dependent sorting algorithms. Measured fluorescence time series of labelled single molecules need to be sorted into ’good molecules’ and ’bad’ molecules before further kinetic and thermodynamic analysis.
Currently, processing, sorting and analysis of the data is mainly done with the help of laboratory specific programs.
Although there are freely available programs for processing smFRET data, they do not offer ’molecular sorting’ or it is purely empirical. Only recently, new approaches came up to solve this problem by means of machine learning. Here, we describe a sound terminology for molecular sorting of smFRET data and present an efficient workflow for manual annotation followed by the training of the ML algorithm. Descriptive statistics of our generated dataset are provided and will serve as the basis for supervised ML-based molecular sorting algorithms yet to be developed.
Ensemble FRET ist eine etablierte Methode zur thermodynamischen Charakterisierung von Biomolekülen, insbesondere von Nukleinsäuren (NA). Hier soll die Faltungsdynamik einer DNA-Doppelhelix (Duplex) untersucht werden, welche mit zwei Fluorophoren Cy3 als Donor und Cy5 als Akzeptor markiert ist. Durch die Zugabe von monovalenten Metallionen wie K(I) oder Na(I) soll der Faltungszustand und damit die Stabilität der DNA mittels FRET temperaturabhängig charakterisiert werden. Unser Ziel ist es einen zuverlässigen NA-FRET Standard für die thermodynamische Charakterisierung von Nukleinsäuren mittels FRET zu etablieren.
This work deals with the construction of a microscope for combined total internal reflection fluorescence (TIRF) and confocal microscopy. It is especially designed for single-molecule fluorescence spectroscopy. The design of the microscope body is based on the miCube (Hohlbein lab, Wageningen University, NL). The excitation and detection pathways were adapted to allow both TIRF and confocal illumination as well as camera and pointdetection for two color-channels to allow single-molecule Förster resonance transfer measurements